Research Article
ISSN: 2475 3432

Meat quality and hematological indices of Oreochromis niloticus fish experimentally exposed to Escherichia coli toxins

Amal M Yacoub*1,Sherifa Mostafa M.2, Sabra, Mona Khaled D. Al-Kourashi3
1(Pollution Lab. / National Institute of Oceanography and Fisheries)
2(Department of Microbiology / Animal Health Research Institute, Dokki, Giza, Egypt)
1,2,3(Biology Dept., Science College, Taif University, KSA)
Corresponding author: Amal M Yacoub
Pollution Lab. / National Institute of Oceanography and Fisheries, Taif University, KSA. E-mail: yacoub_am2006@yahoo.com
Received Date: August 27, 2018 Accepted Date: September 11, 2018 Published Date: September 28, 2018
Citation: Amal M Yacoub et al. (2018), Meat quality and hematological indices of Oreochromis niloticus fish experimentally exposed to Escherichia coli toxins. Int J Biotech & Bioeng. 4:7, 139-148
Copyright: ©2018 Amal Mohammed. Yacoub et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited


Fish of Oreochromis niloticus (Linnaeus, 1758) were treated with different concentrations of Enterotoxigenic Escherichia coli (ETEC) as 103-105, 106 -107 and 109-1010 CFU/ml water. The investigation had conducted to detect experimentally the impact of E. coli toxins on Oreochromis niloticus fish throughout cells blood counts (WBCs, RBCs and Platelets), hemoglobin concentration and histopathological changes of muscles. The values of WBCs, RBCs, Platelets and hemoglobin ranged between (90.70 K/ml -7.10 K/ml), (2.00 m/ml - 0.53 m/ml), (21 K/ml - 3 K/ml) and (8.2 G/dl to 2.50 G/dl) for the hematological indices respectively. The concentration of WBCs, RBCs, Platelets and hemoglobin were lower than control in all the experiment phases. Histopathological changes of O. niloticus treated with E. coli included degeneration, necrosis, atrophy and inflammatory infiltration of WBCs. The results revealed mortality of fish in the highest concentration and high incidence of E. coli toxins in the muscles of O. niloticus induced damage in the muscle tissues and may transfer to the human consumer and cause serious illness.

Keywords:  Experimental Study, Oreochromis Niloticus, Escherichia Coli, Blood Cells Count, Muscles




Introduction:


Fish are susceptible to wide variety of bacterial pathogens especially when the fishes are physiologically unbalanced or nutritionally deficient, or subjected to stressors, i.e. poor water quality and overstocking. Infectious diseases are the main cause of economic losses in aquaculture industry which is negatively impacted by various pathogenic micro-organisms (MOs) such as Escherichia coli (E. coli) (Plumb, 1997).

Escherichia coli is Gram negative rods within the family Enterobacteriaceae, and represents a part of the normal micro-flora of the intestinal tract of human and warm- blooded animals. Due to their high prevalence in the gut, E. coli is used as the preferable indicator to detect and measure fecal contaminate in the assessment of food and water safety. Pathogenic E. coli strains are distinguished by their ability to cause serious illness as a result of their genetic elements for toxins production, adhesion and invasion of the host cells, interference with cell metabolism and tissue destruction (Borgatta et al., 2012). Entero-toxigenic E. coli strains were able to cause diarrhea in adult volunteers (DuPont, et. al., 1971).

The total bacterial count and pathogenic flora coliform of the raw sewage, oxidation pond and culture pond were analyzed. The bacterial load was higher in the gut contents than in skin, gills and muscle. Microbiology of different tissues and gut contents from six different fish species cultured in a sewage-fed pond was studied. The total bacterial count and pathogenic flora coliform of the raw sewage, oxidation pond and culture pond were also analyzed. The bacterial load was higher in the gut contents than in skin, gills and muscle. Detritivorous fish species had a higher bacterial count than the filter feeders. The bacterial load was reduced during the depuration period (20 days in fresh water) of the fishes. The fish-sauce preparation examined revealed the complete elimination of microbes (Balasubramanian et al., 1992). The abundance of coliform bacteria in muscles, gills and intestine of Nile tilapia (Oreochromis niloticus) sampled from different sources. Densities of total aerobic bacteria, total coliform (TC), fecal coliform, (FC) and E. coli were measured from different organs of Nile tilapia sampled from pond, and market using serial dilution and spread plate techniques. Significantly higher density of fecal coliform was detected in the muscle, gill and intestine in Nile tilapia sampled from pond than that of market (Mandel et al., 2009).

A number of 775 apparently healthy and naturally infected fish species; (Tilapia Oreochromis niloticus, catfish, clarias gariepinus and mullets) collected from different areas in Port Said governorate. Collected fishes were subjected to full clinical, bacterial and histopathological examinations. There results revealed that the most common clinical signs of the naturally infected fish were darkening of the skin, hemorrhages in skin, fins, oral cavity and muscles, sloughing of scales with superficial and deep ulceration of the epidermis (El-Refaey, 2013). A study evaluated the hematological changes in Nile tilapia experimentally infected with 1 x 103 and 1 x 106 colony-forming units (CFU)/mL of Enterococcus sp. in the swim bladder. The experiment consisted of four treatments in triplicates: non-injected fish (NI); fish injected with 1 mL of sterile saline solution 0.65% (SAL); fish injected with 1 x 103 and 1 x 106 CFU/mL of Enterococcus diluted in 1 mL sterile saline. Twenty-four hours after injection, the fish were anesthetized and the blood collected. The hematological tests included red blood cell (RBC) and white blood cell (WBC) counts, hematocrit, number of total thrombocytes, and differential counting of WBC. Fish injected with 1 x 106 CFU/mL of Enterococcus showed a higher number of thrombocytes than the other treatments. White blood cell and lymphocyte numbers increased significantly in fish injected with 1 x 106 CFU/mL of Enterococcus when compared to non-injected control. There was significant increase in the number of neutrophils in saline injected fish and reduced number of monocytes after injections with 1 x 106 CFU/mL of Enterococcus. Hematocrit increased in fish injected with 1 x 103 and 1 x 106 CFU/mL of Enterococcus (Martins et al., 2008).

Antioxidant enzymes and the indicators of lipid peroxidation levels in muscles of O. niloticus were found to be significantly increased compared to the reference values in Rosetta branch of River Nile which is exposed to agricultural drainage water, sewage and industrial wastes (Khalil et al., 2017). Several circumstances promote the antioxidant defense response in fish, factors intrinsic to the fish itself such as: age, reproductive, metabolic status of fish and environmental conditions, that include food availability, oxygen level, temperature of water, salinity and photoperiod, toxins present in the water or pathologies, can either fortify or weaken antioxidant defenses (Melegaria et al. 2013).

Nile tilapia (Oreochromis niloticus) belongs to the family of Cichlidae and is known to the ancient Egyptian for more than five thousand years ago. It was common in natural fisheries in Africa, such as Nile River and freshwater lakes. The original home of tilapia is the content of Africa. Because of its importance, it has been introduced and spread in the world where there is warm water appropriate for their growth and reproduction especially in Southeast Asian countries (Dey et al., 2000). Most tilapia species are suitable for aquaculture. The growth rate varies depending on the nature of the food. It feed mainly on phytoplankton, algae, aquatic plants, remaining of decaying organic material and insect larvae (Tharwat, 2013). The fish is a good source of proteins for human. Untreated sewage water contains intestinal pathogens as E. coli. Some fish breeders use untreated sewage water for growing fish. So this research had conducted to detect the experimentally impact of E. coli toxins on Oreochromis niloticus fish understudy throughout hematological tests of cells blood count and histopathogical changes of muscles to confirm the hazardous effect of using sewage water on fish meat quality and health status.

Material and Methods


Oreochromis niloticus fish were bought from a fish farm then transferred to water aquaria for acclimatization. Weights of tilapia fish were (200-250 gm.)
Tap water was used in the experimental aquaria. The physicochemical characters of tap water were measured. The results revealed that tap water was free of bacterial pollution, nitrite and ammonia (Table 1). Gotten isolation of ETEC (Enterotoxigenic Escherichia coli) from authorized laboratory and prepared the concentration for experiment, and exposed the fish to different concentrations (Mc Farland, 2009).
All sinks were filled with de-chlorinated water at room temperature. O. niloticus were exposed to control and three consecutive doses of ETEC (ETEC concentrations were 103-105/ml water, 106-107/ml water and 109-1010/ml). Samples of fish and water on control and consecutive periods were collected according to plan at (1st, 3rd, 5th, 7th and 9th days) of experiment.
ETEC were isolated and classified from the blood, skin and muscles of under experiment fish and water (Cline. Lab., 2011; CDC, 2013). The collected data was subjected to statistical analysis using one- way Analysis of Variance (ANOVA).

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Table 1:Physicochemical characters and microbial analysis of tap water where fish survived in the bioassay aquaria

Hematological methods:
Blood samples were taken from the caudal vein of the fish using a heparinized syringe. Cells blood count (CBC) were measured by cell from Abbott company, USA. Blood samples were used to estimate White blood cells (WBC), Red blood cells (RBC) and Platelets count and hemoglobin concentration by the method of Dumas and Biggs (1972).
Histological methods:

Nile tilapia (O. niloticus) fish were dissected and specimens of muscles were removed, washed in saline solution and prepared for histological study. Muscles specimens were fixed in 10% formalin for 48 hours. After fixation, fish muscles were dehydrated in ascending grades of ethanol, cleared in pure xylene then embedded in paraffin wax. The paraffin wax blocks were serially sectioned with microtome at 5 micrometers. Finally, the sections were mounted on glass slides, stained with hematoxylin and eosin (Bernet et al., 1999). The stained sections were examined and photographed with OMAX light microscope with USB digital build in camera.

Results


Table (2) showed the incidence of re-isolation of ETEC during the experiments phases, ETEC were 00% from control during all phases of experiment. At 1st phase ETEC re-isolation from water was (25, 25, 50, 75 and 75%) at 1st, 3rd, 5th, 7th and 9th days respectively. ETEC re- isolation from skin and muscles were (25, 25, 25, 50 and 50%). At 2nd phase the ETEC re-isolation from water was (25, 50, 50, 75 and 100%) and in skin and muscles as (25, 50, 75, 75 and 100%) at 1st, 3rd, 5th, 7th and 9th days respectively. At 3rd phase, the fish did not survive i.e. all fish died with ETEC concentration after the 5th day of the phase. The ETEC re-isolation from water was (25, 50 and 75%) at 1st, 3rd and 5th days respectively. ETEC re-isolation from skin and muscles was (50, 75 and 75%) at 1st, 3rd and 5th days respectively. ETEC were zero in blood in all phases of experiment.

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Table 2: Incidence of re-isolation of ETEC during the experiment phases

White Blood cells count:


The maximum value of WBCs in concentration (103 - 105 CFU / ml) was 53.5 K/ml in the 5th day and the minimum value was 30.0 K/ml in the 1st day .The mean ± standard deviation was 40.84 ±11.66.
The maximum value of WBCs in concentration (106- 107 CFU / ml) was 90.70 K/ml in the 5th day and the minimum value was 7.10 K/ml in the 3rd day .The mean ± standard deviation was 43.38 ±32.37.
The experiment only lasted for five days because of the death of the fish in concentration (109 – 1010 CFU /ml), the maximum value of WBCs was 55.10 K/ml in the 3rd day and the minimum value was 31.60 K/ml in the 5th day. The mean ± standard deviation was 41.16 ±12.34.
The values of WBCs count in the experiment ranged from the highest value 90.70 K/ml in the 5th day in concentration (106- 107 CFU /ml) and the lowest value 7.10 K/ml in the 3rd day in the same concentration. The control was 100 K/ml.

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(Table and diaphragm 3) showing White Blood Cells count of Oreochromis niloticus treated with different concentration of ETEC.

Red Blood cells count:

The maximum value of RBCs in concentration (103 - 105 CFU/ ml) was 2.00 m/ml in the 5th day and the minimum value was 1.29 m/ml in the 1st day .The mean ± standard deviation was 1.56 ± 0.34.
The maximum value of RBCs in concentration (106 - 107 CFU / ml) was 1.60 m/ml in the 5th day and the minimum value was 0.53 m/ml in the 3rd day. The mean ± standard deviation was 1.28 ± 0.44.
The maximum value of RBCs in concentration (109 – 1010 CFU /ml) was 1.65 m/ml in the 1st day and the minimum value was 1.24 m/ml in the 5th day. The mean ± standard deviation was 1.48 ± 0.21.
The values of RBCs count in the experiment ranged from the highest value 2.00 m/ml in the 5th day in concentration ( 103- 105 CFU /ml) and the lowest value 0.53 m/ml in the 3rd day in the concentration (106 - 107 CFU /ml ). The control was 2.26 m/ml.

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(Table and diaphragm 4) showing Red Blood Cells count of Oreochromis niloticus treated with different concentration of ETEC

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(Table and diaphragm 4) showing Red Blood Cells count of Oreochromis niloticus treated with different concentration of ETEC

Platelets count:

The maximum value of Platelets in concentration (103 - 105 CFU/ ml) was 21 K/ml in the 7th day and the minimum value was 9 K/ml in the 5th day .The mean ± standard deviation was 16.8±4.60.
The maximum value of Platelets in concentration (106 - 107 CFU / ml) was 17 K/ml in the 7th day and the minimum value was 3 K/ml in the 3rd day .The mean ± standard deviation was 9.8±5.97.
The maximum value of Platelets in concentration (109 – 1010 CFU/ml) was 17 K/ml in the 1st& 5th days and the minimum value was 13 K/ml in the 3rd day. The mean ± standard deviation was 5.66±2.30.
The values of Platelets count in the experiment ranged from the highest value 21 K/ml in the 7th day in concentration ( 103 - 105 CFU/ml ) and the lowest value 3 K/ml in the 3rd day in the concentration (106 - 107 CFU /ml ). The control was 30 K/ml.

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(Table and diaphragm 4) showing Red Blood Cells count of Oreochromis niloticus treated with different concentration of ETEC

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(Table and diaphragm 5) showing Platelets count of Oreochromis niloticus treated with different concentration of ETEC.

Hemoglobin concentration:

The maximum value of Hemoglobin in concentration (103 - 105 CFU / ml) was 8.2 G/dl in the 1st day and the minimum value was 6.3 G/dl in the 9th day .The mean ± standard deviation was 7.36±0.82.
The maximum value of Hemoglobin in concentration (106- 107 CFU/ ml) was 7.10 G/dl in the 5th day and the minimum value was 2.50 G/dl in the 3rd day .The mean ± standard deviation was 5.76±1.85.
The maximum value of Hemoglobin in concentration (109-1010 CFU /ml) was 7.70 G/dl in the 1st day and the minimum value was 6.30 G/dl in the 5th day. The mean ± standard deviation was 6.96±0.70.
The values of Hemoglobin concentration in the experiment ranged from the highest value 8.2 G/dl in the 1st day in concentration (103- 105 CFU /ml ) and the lowest value 2.50 G/dl in the 3rd day in the concentration (106- 107 CFU / ml ). The control was 9.3 G/dl.

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(Table and diaphragm 4) showing Red Blood Cells count of Oreochromis niloticus treated with different concentration of ETEC

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(Table and diaphragm 6) showing Hemoglobin concentration of Oreochromis niloticus treated with different concentration of ETEC.

Histopathological changes of muscles of Oreochromis niloticus experimentally treated with Escherichia coli:

The striated muscle cell of the body musculature, or the skeletal muscles, is multinucleated. The nuclei lie just beneath the membranous sarcolemma that sheathes the cell. Each cell contains several longitudinal myofibrils, each of which is comprised of several myofilaments. The myofibrils can be seen by light microscopy, but myofilaments are visible only by electron microscopy. The structure of normal muscles in (Fig.1) The muscles of Oreochromis niloticus treated with concentration (103-105 CFU /ml) of Escherichia coli demonstrated degeneration of myotomes in the 1st day and 9th day (Fig.2, 3and8).In the 3rd day, necrosis of mytomes appeared (Fig.4and6).In the 3rd day, microscopical examination showed fragmentation of mytomes (Fig.5) and atrophy of myotomes in the 5th day (Fig.7). The muscles of Oreochromis niloticus treated with concentration (106-107 CFU /ml) of Escherichia coli demonstrated fragmentation of collagen bundles in the 1st day (Fig.9). In the 3rd day, necrosis of myotomes was observed in (Fig.10 and11).In the 5th day and 9th day, atrophy of myotomes appeared in (Fig.12 and14) and in the 7th day and 9th day, degeneration of myotomes was shown in (Fig.13 and14). The muscles of Oreochromis niloticus treated with concentration (109-1010 CFU /ml) of Escherichia coli demonstrated necrosis of myotomes in the 1st, 3rd and 5th day (Fig.15, 16, 17,19 and20). In the 3rd day, atrophy of myotomes and pyknotic of nuclei were noticed in (Fig.18) and inflammatory infiltration of WBCs in the 5th day (Fig.21).

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(Fig.1)& (Fig.2): Normal structure of muscles of Oreochromis niloticus fish from the control group, showing the myotomes (M).
& L.S. of Oreochromis niloticus muscles treated with concentration (103_105 CFU /ml) of ETEC in the 1st day of exposure, showing degeneration of myotomes (D).

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(Fig.3) & (Fig.4): L.S. of Oreochromis niloticus muscles treated with concentration (103_105 CFU /ml) of ETEC in the 1st day of exposure, showing degeneration and infiltration of WBCs (arrow)
L.S. of Oreochromis niloticus muscles treated with concentration (103_105 CFU /ml) of ETEC in the 3rd day of exposure, showing vacuolar necrosis (V) of myotomes.

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(Fig.5)& (Fig.6): L.S. of Oreochromis niloticus muscles treated with concentration (103_105 CFU /ml) of ETEC in the 3rd day of exposure, showing fragmentation of myotomes (arrow).
L.S. of Oreochromis niloticus muscles treated with concentration (103_105 CFU /ml) of ETEC in the 3rd day of exposure, showing necrosis (N) of myotomes.

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(Fig.7) & (Fig.8): L.S. Oreochromis niloticus muscles treated with concentration (103_105 CFU /ml) of ETEC in the 5th day of exposure, showing atrophy of myotomes (A).
L.S. of Oreochromis niloticus muscles treated with concentration (103_105 CFU /ml) of ETEC in the 9th day of exposure, showing degeneration and rupture of myotomes (arrow).

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(Fig.9) & (Fig.10): L.S. of Oreochromis niloticus muscles treated with concentration (106_107 CFU /ml) of ETEC in the 1st day of exposure, showing fragmentation of collagen bundles (arrow).
L.S. of Oreochromis niloticus muscles treated with concentration (106_107 CFU /ml) of ETEC in the 3rdday of exposure, showing necrosis(N) and atrophy (A)of myotomes (arrow).

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(Fig.11) & (Fig.12): L.S. of Oreochromis niloticus muscles treated with concentration (106_107 CFU /ml) of ETEC in the 3rd day of exposure, showing vacuolar necrosis (V) of myotomes .
L.S. of Oreochromis niloticus muscles treated with concentration (106_107 CFU /ml) of ETEC in the 5thday of exposure, showing atrophy of myotomes (arrow).

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(Fig.13) & (Fig.14): L.S. of Oreochromis niloticus muscles treated with concentration (106_107 CFU /ml) of ETEC in the 7th day of exposure, showing degeneration (D) of myotomes.
L.S. of Oreochromis niloticus muscles treated with concentration (106_107 CFU /ml) of ETEC in the 9th day of exposure, showing degeneration (D) and atrophy (A) of myotomes.

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(Fig.15) & (Fig.16):L.S. of Oreochromis niloticus muscles treated with concentration (109_1010 CFU /ml) of ETEC in the 1st day of exposure, showing necrosis (N) of myotomes.
L.S. of Oreochromis niloticus muscles treated with concentration (109_1010 CFU /ml) of ETEC in the 1st day of exposure, showing severe necrosis (N). The myotomes lost their normal architecture.

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(Fig.17) & (Fig.18):L.S. of Oreochromis niloticus muscles treated with concentration (109_1010 CFU /ml) of ETEC in the 3rd day of exposure, showing necrosis of myotomes (N).
L.S. of Oreochromis niloticus muscles treated with concentration (109_1010 CFU /ml ) of ETEC in the 3rd day of exposure, showing atrophy of mytomes(A) and pyknotic(P) nuclei.

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(Fig.19) & (Fig.20):L.S. of Oreochromis niloticus muscles treated with concentration (109_1010 CFU /ml) of ETEC in the 5th day of exposure, showing necrosis of myotomes (N).The muscles lot their normal architecture.
L.S. of Oreochromis niloticus muscles treated with concentration (109_1010 CFU /ml) of ETEC in the 5th day of exposure, showing vacuolar necrosis of myotomes (N).

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(Fig.21): L.S. of Oreochromis niloticus muscles treated with concentration (109_1010 CFU /ml) of ETEC in the 5th day of exposure, showing inflammatory infiltration of WBCs (arrow).

Discussion


Tilapia is one of the most cultivated freshwater fish worldwide. The variation degree on the hematological response is an important tool to fish health diagnosis and may vary according to stressors stimulus, treatment, parasitic or infectious diseases (Silveira- Coffigney et al.,2004; Chen et al.,2004 and Martins et al., 2004). In this study, the values of WBCs, RBCs, Platelets (thrombocytes) and hemoglobin were lower than control. This assay showed that total number of WBCs and thrombocytes were affected by the bacterial injection. An interesting result was related to the decreased number of monocytes (WBCs) in 1 x 106 Enterococcus/ mL injected fish (Martins et al., 2008). Possibly these cells are being recruited to the lesion site as commented by Matushima and Mariano (1996) and Martins et al. (2006).

Decreased RBCs count and hemoglobin indicate that erythrocytes are being destroyed with the infection. Decreased RBCs and hemoglobin in chum salmon infected with V. anguilarum, in rainbow trout infected with Aeromonas/ Streptoccus and in Cichlid fish with epizotic ulcerative syndrome were previously reported ( Harbell et al., 1979; Barham et al., 1980 and Pathiratne and Rajapakshe, 1998). Contrarily to the results of this study, Martins et al. (2008) reported that, the injection of Enterococcus was found to incite more production of platelets (thrombocytes) in the circulating blood. Little studies relate the hematological parameters to bacterial experimental infection. For example, in rainbow trout (Oncorhinchusmykiss) with ulcerous dermatitis (Rehulka, 1998) in rainbow trout experimentally infected with Aeromonassobria and A. caviae (Rehulka, 2002) in carp (Cyprinuscarpio) experimentally infected with Streptoccusiniae (Chen et al., 2004). On the other hand, an increase in WBCs was observed by Haney et al. (1992) in chum salmon (Oncorhynchusketa) with erythrocytic necrosis virus. The highest red blood cells count (RBCs), Hemoglobin (Hb) and hematocrite (Hct) were recorded by Hassanien et al., (2017) for fish fed the diet supplemented with Saccharomyces cerevisiae.

The muscles of O. niloticus treated with E. coli showed degeneration, necrosis and atrophy of mytomes in addition to inflammatory infiltrationof WBCs and pyknotic nuclei of myocytes. Lazar et al. (2011) studied bacterial hemorrhagic speticimia of ciprinides caused by Aeromonas sp. bacteria, in association to Pseudomonas sp. bacteria. The examination of somatic muscles showed interstitial edematous infiltration. Weisman and Miller (2006) found myocyte degeneration with cytoplasmic vacuoles, loss of cross striation, fragmentation and infiltration of inflammatory cells in agreement with our results. Monocytes degeneration may be attributed to vitamin E deficiency, such as may occur following ingestion of rancid food (Smith, 1979; Holliman and Shouthgate, 1986 &Tacon, 1996). Vitamin E is an antioxidant that helps prevent peroxidation of adipose tissue (Comporti, 1993).

In conclusion, this study concerned with the problem of sewage discharge in water bodies and using it in fish farms and its threat on public health. The results of the present investigation revealed that treatment of Oreochromis niloticus fish with Escherichia coli decreased blood cells count and induced pathological changes in muscles and finally caused mortality in the highest concentration. It is recommended to prevent fishing in seas or rivers polluted with sewage or usage of sanitation on farms.

Acknowledgement:


The authors like to extend their appreciation to the owners of Ba Ghanem fish farm, Jeddah KSA for their perfect transport of Nile Tilapia fish samples.


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